1. Field of the Invention
This invention lies in the fields of phosphorylated peptides and the fractionation of peptides in general.
2. Background of the Invention
Analyses of phosphorylated peptides are used in studies of protein functions and biological systems. Protein phosphorylation occurs during posttranslational modification of proteins and in many cases is crucial to the preservation and regulation of protein structure and function. Enzymatically catalyzed phosphorylation and dephosphorylation are important regulatory functions of living cells, contributing to cell proliferation, development, and differentiation, signal transduction, nerve activity, organization of the cytoskeleton, programmed cell death, and gene expression. The determination of phosphorylation sites, performed by digestion of proteins and analysis of the resulting peptides, is thus an important factor in understanding complex biological systems and the development of diseases. Studies of phosphorylated proteins and peptides are made difficult however by the relatively small amounts of these species in biological fluids. Various methods have therefore been developed to achieve selective enrichment of phosphorylated species.
One widely reported means of enrichment of phosphorylated proteins and peptides is immobilized metal chelate affinity chromatography (IMAC). This technique uses separation media that have been surface-modified by the addition of metal ions through chelating agents, and the isolation of phosphopeptides occurs as the result of a strong interaction between the immobilized metal ions and the phosphate groups on the peptides. Various amino acids, however, also display an interaction with the metal ions by acting as electron donors. This competes with the phosphorylated species, and limits the selectivity of the separation. Another difficulty with IMAC is that elution of the bound phosphopeptides is not quantitative, and this limits the efficiency of the separation. High yield and high purity for phosphopeptide enrichment thus remain elusive.
Of further potential relevance to the background of this invention are disclosures of the use of ceramic hydroxypatite (CHT) as a chromatographic separation medium. Disclosures of the use of CHT to purify monoclonal antibodies appear in the review article of Gagnon, P., “Monoclonal antibody purification with hydroxyapatite,” New Biotechnol. 25(5): 287-293 (2009); disclosures of CHT to isolate DNA are reported by Ivanov, I., et al., “Purification of spin-labeled DNA by hydroxyapatite chromatography,” J. Chromatog. 260: 177-183 (1983); and disclosures of CHT to isolate RNA are reported by Kothari, R. M., et al., “RNA Fractionation on hydroxyapatite columns,” J. Chromatog. 98(2): 449-475 (1974). A disclosure of phosphorylated proteins from their non-phosphorylated forms on CHT appears in Schmidt, S. R., et al., “Current methods for phosphoprotein isolation and enrichment,” J. Chromatog. B 849: 154-162 (2006).